Detection and amplification of ligands

ABSTRACT

A system for the detection of ligands comprising at least one receptor and an amplification mechanism coupled to the receptor wherein an amplified signal is produced as a result of receptor binding a ligand. Examples of suitable amplification mechanisms include antibody-embedded liquid crystalline materials; use of alpha-2-macroglobulin to encage an enzyme, whereby the enzyme is separated from its substrate by an receptor; and a receptor engineered to inhibit the active of site of an enzyme only in the absence of a ligand. Also provided are methods for the automatic detection of ligands.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/726,134, filedDec. 1, 2003, now U.S. Pat. No. 7,160,736 which issued Jan. 9, 2007,which is a continuation of application Ser. No. 09/821,396 filed on Mar.29, 2001, now abandoned, which is a continuation-in-part of U.S. Ser.No. 09/633,327 filed Aug. 7, 2000, now abandoned, which is acontinuation of U.S. Ser. No. 09/095,196 filed Jun. 10, 1998, now U.S.Pat. No. 6,171,802, each of which are hereby incorporated herein byreference.

TECHNICAL FIELD OF THE INVENTION

The present invention generally relates to the detection of a ligand bya receptor. More specifically, the present invention relates to highlyspecific receptors and the incorporation of these receptors into anamplification mechanism for the rapid and automatic detection of theligand, particularly pathogens and/or their toxins.

BACKGROUND OF THE INVENTION

The detection of a ligand by a receptor (for example, detection of apathogenic agent such as a microbe or toxin by an antibody; or detectionof an antibody in blood by another antibody; or binding of a chemicaltoxin, such as nerve gas, to its receptor) is important in the diagnosisand treatment of individuals exposed to disease-causing agents. Earlydetection of pathogenic agents can be a great benefit in either diseaseprophylaxis or therapy before symptoms appear or worsen.

Every species, strain or toxin of a microbe contains unique surfaceligands. Using molecular engineering and/or immunological techniques,receptor molecules, such as antibodies, can be isolated that will bindto these ligands with high specificity. Methods have also been developedwhere receptors, such as antibodies, are linked to a signaling mechanismthat is activated upon binding. Heretofore, however, no system has beendeveloped that can quickly and automatically detect and amplify areceptor signal coming from the binding of a single or a low number ofligands. Such a system is imperative for rapid and accurate earlydetection of ligands.

Most available diagnostic tests are antibody based, and can be used todetect either a disease-causing agent or a biologic product produced bythe patient in response to the agent. There are currently threeprevailing methods of antibody production for recognition of ligands(antigens): polyclonal antibody production in whole animals withrecognition for multiple epitopes, monoclonal antibody production intransformed cell lines with recognition for a single epitope (afterscreening), and molecularly engineered phage displayed antibodyproduction in bacteria with recognition of a single epitope (afterscreening). Each of these receptor systems is capable of binding andidentifying a ligand, but the sensitivity of each is limited by theparticular immunoassay detection system to which it is interfaced.

Immunoassays, such as enzyme-linked immunosorbent assay (ELISA), enzymeimmunoassay (EIA), and radioimmunoassay (RIA), are well known for thedetection of antigens. The basic principle in many of these assays isthat an enzyme-, chromogen-, fluorogen-, or radionucleotide-conjugatedantibody permits antigen detection upon antibody binding. In order forthis interaction to be detected as a color, fluorescence orradioactivity change, significant numbers of antibodies must be bound toa correspondingly large number of antigen epitopes.

Thus, there is a need for a system that rapidly, reliably, andautomatically detects ligands, especially when present in very smallquantities and consequently provides a measurable signal.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a systemthat will detect a ligand with high sensitivity and high specificity.

It is another object of the present invention to provide a system thatwill amplify a signal produced by the binding of a ligand to a receptor.

It is another object of the present invention to provide a caged enzymeamplification mechanism.

It is yet a further object of the present invention to provide a systemthat will activate an enzyme inactivated by a bound receptor.

It is still a further object of the present invention to provide asystem that will distort a liquid crystal array upon the binding of aligand to a receptor.

It is still yet a further object of the present invention to provide adetection device that will continuously monitor the environment or thebody and signal its possessor when a ligand is present.

In general, the present invention provides a system for the detectionand amplification of ligands, such as pathogenic agents, comprising atleast one receptor and an amplification mechanism coupled to thatreceptor, wherein an amplified signal is produced as a result of thereceptor binding the ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the lamellar structure of alyotropic liquid crystal formed by alternating layers of water andbiphilic molecules.

FIG. 1B is a schematic representation of the amplification mechanismwith a receptor inserted into the lyotropic liquid crystal.

FIG. 1C is a schematic representation of the amplification mechanismwith the specific ligand bound to its receptor causing deformation ofthe liquid crystal and alteration of the transmission of polarizedlight.

FIG. 2A is a schematic representation of a caged enzyme amplificationmechanism in which one or more receptors blocks the channel leading tothe enzyme in the absence of a ligand.

FIG. 2B is schematic representation of a caged enzyme amplificationmechanism in which a ligand binds to the receptor causing distortion ofthe linker region and the unblocking of the channel leading to theenzyme.

FIG. 2C is a schematic representation of a caged enzyme antibodyamplification mechanism in which, once activated by ligand binding toreceptor, the channel leading to the enzyme remains open and multiplesubstrates can interact with the enzyme.

FIG. 3A is a schematic representation of an enzyme inactivated by amolecularly-engineered receptor.

FIG. 3B is a schematic representation of the binding of a ligand to themolecularly-engineered receptor causing dissociation of the receptorfrom the enzyme, exposing the active site of the enzyme to thesubstrate.

FIG. 3C is a schematic representation of substrate conversion by theenzyme, after picogram levels of ligand bind to the receptor.

FIG. 4A is a photograph showing the birefringence of polarized lightthrough a liquid crystal-receptor array in response to binding of aspecific ligand to the receptor.

FIG. 4B is a photograph showing the absence of birefringence ofpolarized light through a liquid crystal-receptor array in a reactionmixture containing liquid crystal, receptor and PBS.

FIG. 4C is a photograph showing the absence of birefringence ofpolarized light through a liquid crystal-receptor array in a reactionmixture containing liquid crystal, receptor and an irrelevant ligand.

FIG. 5 is a graph showing the quantative analysis of the birefringenceof polarized light through an liquid crystal-receptor array of theligand detection system for selective receptor-ligand binding, ascompared to non-selective receptor-ligand binding and backgroundreceptor-ligand binding.

FIG. 6A is a graph showing enzymatic activity of elution fractionscontaining an alpha-2-macroglobulin-trypsin complex.

FIG. 6B is a graph showing the protein concentration (ug/ml) of elutionfractions containing an alpha-2-macroglobulin-trypsin complex.

FIG. 6C is a graph showing the effect of the concentration of cagedtrypsin on detectable enzymatic activity over time (min.).

FIG. 6D is a graph showing the effect of the concentration of cagedthrombin on detectable enzymatic activity over time (min.).

FIG. 6E is a graph showing the stability of caged trypsin over time(days).

FIG. 7A is a graph showing luciferase activity (Relative Light Units)over time (sec.).

FIG. 7B is a graph showing luciferase activity (Relative Light Units) asa function of luciferase concentration (pg/ul).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the present invention, ligand-specific receptors are interfaced withan amplification mechanism such that a receptor-ligand interactionchanges the conformation of the receptor and produces a signal.Amplification preferably occurs through a calorimetric, fluorometric orbirefringent shift that can be photometrically detected. The detectedsignal may then be electronically amplified to automate the system.

Ligand Detection Component

Any receptor, such as antibodies or biologic/biologically engineeredreceptors for ligands, can be incorporated into the device as long asbinding of the ligand to the receptor causes a detectable distortion ofthe receptor. For example, any type of monospecific antibody(polyclonal, monoclonal, or phage displayed) can effectively function asa receptor, and thus each of those antibody types will be described inthe following paragraphs. Although phage-displayed antibodies can beexpeditiously modified for identification of new ligands and are used asreceptor examples in this patent application, any physically-distortablereceptor-ligand interaction is appropriate for the detection component.

Polyclonal antibodies: Antibody-based antigen detection has beenexploited for several decades. Injection of a purified ligand (antigen)into a host animal stimulates the immune system to produce an array ofantibodies against various reactive sites on the antigen. Since severallymphocytes are responding to different antigenic epitopes, amulti-specific antibody cocktail (polyclonal) is created and can bepurified for antigen detection.

Monoclonal antibodies: Antibody-producing spleen cells (B lymphocytes)are fused with immortalized myeloma cells to create hybridomas whichprovide nearly infinite quantities of antibody with a single, definedspecificity. Interstrain and even interspecies hybrids of these‘monoclonal’ antibodies can be generated through genetic engineeringtechniques. These highly specific antibodies have significanttherapeutic potential, as evidenced by the U.S. Food and DrugAdministration's approval of the use of mouse-human chimeric antibodiesfor treatment of selected diseases.

Phage-displayed mono-specific antibodies: Phage-displayed techniqueswill be used to isolate single chain chimeric antibodies to variouspathogenic agents. The genomic DNA of the B lymphocyte contains the codeto produce an antibody to virtually all possible ligands (antigens). Ina phage displayed antibody system (PDA), DNA encoding a single chainchimera of the native antibody's hypervariable ligand-binding region issynthesized by joining DNA encoding an antibody heavy chain and DNAencoding an antibody light chain and inserting therebetween DNA encodinga linker region. The desired amino acid sequence of the linker regiondepends on the characteristics required for any given amplificationmechanism. The linker region may have to be able to interact and/or bondto a protein or other substance. Therefore, the polypeptide sequence mayhave to have, for example, a particular conformation, specificallyplaced functional groups to induce ionic or hydrogen bonds, or ahydrophobicity that is compatible with the amplification mechanism.Regardless of the type of amplification mechanism, however, the linkerregion plays a critical role in interfacing the amplification mechanismto the receptor.

The DNA, preferably human or mouse, encoding the single chain chimericantibody is cloned into a bacteriophage (phage) vector using well-knowntechniques (Marks et al., J. Mol. Bio. Vol. 222:581 (1991); Griffiths etal., EMBO J. 12:725 (1993); and Winters et al., Ann. Rev. Immunol.12:433 (1994)), incorporated herein by reference. The single chainchimeric antibodies then become displayed on the surface of afilamentous phage with the hypervariable antigen-binding site extendedoutward.

After the addition of ligands, phage that are reactive againstnon-targeted ligands are subtracted from the phage library using knowntechniques (Marks et al., J. Mol. Bio. Vol. 222:581 (1991); Griffiths etal., EMBO J. 12:725 (1993); and Winters et al., Ann. Rev. Immunol.12:433 (1994)), incorporated herein by reference. The remaining phageare reacted with their specific ligand and phage reactive with thatspecific ligand eluted. Each of these phage are then isolated andexpressed in a bacterial host, such as Escherichia coli (E. coli) toproduce a large quantity of phage containing the desiredsurface-displayed antibody. Each of the aforementioned methods relatingto synthesizing and cloning DNA, subtracting phages, isolating andexpressing phages and recovering viral DNA are well known and fullydescribed by Marks et al., J. Mol. Biol. (1991); Griffiths et al., EMBOJ. 12:725 (1993); and Winters et al., Ann. Rev. Immunol. 12:433 (1994),all of which are incorporated herein by reference.

Amplification Component

Any mechanism that permits detection of ligand-receptor complexformation functions as an amplifier and can be incorporated into thedevice. Three amplification mechanisms are proposed. First, a liquidcrystal will amplify the distortion caused when a ligand binds to areceptor. Second, an enzyme will be placed in a biologic cage and areceptor will be attached to the biologic cage to preventenzyme-substrate interaction. Ligand attachment to the receptor willopen a substrate channel, resulting in enzyme-substrate interaction, andthus permitting detectable levels of reaction product. Third, the linkerregion of a receptor, such as a phage-displayed antibody, will beengineered to bind and inhibit the active site of an enzyme.Dissociation of the receptor-enzyme complex occurs upon formation of anreceptor-ligand complex, such as an antigen-antibody complex, andresults in activation of the enzyme and generation of product.

Liquid Crystal: A liquid crystal is a state of matter in which moleculesexhibit some orientational order but little positional order. Thisintermediate ordering places liquid crystals between solids (whichpossess both positional and orientational order) and isotropic fluids(which exhibit no long-range order). Solid crystal or isotropic fluidcan be caused to transition into a liquid crystal by changingtemperature (creating a thermotropic liquid crystal) or by using anappropriate diluting solvent to change the concentration of solidcrystal (creating a lyotropic liquid crystal). Lyotropic liquid crystalswill be used for our amplification system.

As seen in FIG. 1A, most lyotropic liquid crystals, designated generallyby the numeral 1, are formed using water 2 as a solvent for biphilicmolecules 3, for example, molecules which possess polar (hydrophilic)aliphatic parts 4 and a polar (hydrophobic) aliphatic parts 5. Whenwater 2 is added to a biphilic molecule 3, such as the cationicsurfactant cetylpiridinium chloride [C₂₁H₃₈ClN], a bilayer 6 forms asthe hydrophobic regions coalesce to minimize interaction with water 2while enhancing the polar component's interaction with water. Theconcentration and geometry of the specific molecule define thesupramolecular crystalline order of the liquid crystal. The moleculescan aggregate into lamellae as well as disk-like or rod-like micellesthat form a nematic or cholesteric phase. The C₂₁H₃₈ClN forms a lamellaof alternating layers of water and biphilic molecules. An orientationalorder is created by the alternating layers of water and biphilicmolecules and thus the liquid crystal is opaque (exhibits opticalanisotropy) to polarized light 7 provided by a light source locatedperpendicular to the plane of the liquid crystal.

Most biologic receptors possess both hydrophilic and hydrophobic regionsand thus readily incorporate into biphilic lyotropic liquid crystals.Additionally, the inactivated receptors do not destroy the opticalanisotropy of the liquid crystal and therefore, the receptor-enrichedliquid crystal remains opaque to polarized light (FIG. 1B). However,optical anisotropy is disrupted when receptor conformation shifts asduring the formation of the receptor-ligand complex (FIG. 1C). Theelasticity of the liquid crystal enhances the local distortions in thevicinity of the receptor-ligand complex, and expands it to an opticallydetectable, supramicron scale. Biologic materials can be detected on thesurface of thermotropic liquid crystals (V. K. Gupta et al., Science279:277-2080, 1998). However, lyotropic liquid crystals readilyincorporate ligand-specific receptors, and are thus clearly superior fordetection of biologic molecules.

Configurations of Ligand Detection System

By way of example, one envisioned application of the present inventionis in a multiwell system. Each well of the system would contain PDAs toa specific ligand, such as a pathogenic microbe, interfaced with anamplification mechanism of the present invention. When the microbialagent interacts with the antibody, the resulting antibody distortiontriggers the amplification mechanism. Preferably, the amplified signalis then transduced into a perceptible signal. Accordingly, it isenvisioned that such a system could be placed in a physician's office,and be used in routine diagnostic procedures. Alternatively, such asystem could be placed on or near soldiers in battle, and the inventionused to alert the soldiers to the presence of a toxic agent. It isfurther envisioned that a multiwell system, although it can be used withother embodiments, such as the luciferase or the caged enzyme describedhereinbelow, is preferably used in conjunction with the liquid crystalembodiment described herein.

Thus, in one embodiment of the present invention, shown schematically inFIGS. 1B and 1C, a lyotropic liquid crystalline material is used as anamplification mechanism. As shown in FIG. 1B, the device consists of alight source 10, an initial polarizer 12, with the direction ofpolarization in the plane of the figure, a pathogen detection system 14a, comprising monospecific antibodies 14 b embedded in biphilic,lyotropic liquid crystalline material 14 c, a secondary polarizer 16,with the direction of polarization perpendicular to the plane of thefigure, and a photodetector 18.

In operation, the initial polarizer 12 organizes a light stream 22 thatis linearly polarized in the plane of the figure. The optical axis 20 ofthe inactivated device is perpendicular to the pathogen detection system14 a, and thus no birefringence of the transluminating linearlypolarized light stream 22 occurs. Since the polarization direction ofthe secondary polarizer 16 is perpendicular to the transluminatinglinearly polarized light 22, the secondary polarizer prevents light fromreaching the photodetector 18.

Binding of a ligand 24, such as a microbe, to the receptor 14 b, such asan antibody, distorts the liquid crystal 14 c, induces birefringence andthus causes the generation of detectable light. This activation processis illustrated in FIG. 1C. The receptor (antibody) 14 b is embedded inthe biphilic, lyotropic liquid crystal 14 c. The spacial distortioncaused by the formation of the antigen-antibody complex is transmittedto the contiguous liquid crystal 14 c. The elastic characteristics ofthe liquid crystal permit the distortion to be transmitted over a regionmuch larger than the size of the receptor-ligand complex. This allowsthe use of the standard optical phenomenon of birefringence to detectdistortions caused by the receptor-ligand complex. The altered liquidcrystalline order tilts the optical axis 20 and induces birefringence.In other words, the incident polarized light 22 gives rise to tworefracted light waves: the ordinary wave and the extraordinary wave withthe mutually orthogonal polarizations (see, Max Born and E. Wolf.,Principals of Optics, Sixth edition, Pergaman Press, Oxford, 1980),incorporated herein by reference. Thus, there is a portion of light 26in which the optic vibrates in the direction of the secondary polarizer16. The secondary polarizer (analyzer) 16 allows this portion of thelight to pass to the photodetector 18. The detected change oramplification in light intensity can be transduced electronically into aperceptible signal.

In another embodiment of the present invention, shown schematically inFIGS. 2A-2C, enzyme-substrate interactions are exploited foramplification. With reference to FIG. 2A, an enzyme 30 is entrappedwithin a cage 32 to prevent premature interaction of enzyme 30 and asubstrate 34. Substrate 34 can potentially gain access to enzyme 30through one or more channels 36 transecting cage 32. Channel 36,however, is blocked as indicated by arrow 37, by one or more receptorshaving dual binding capacities (diabodies) 38 in the absence of apathogenic agent.

To produce a diabody, two antibodies are coupled, preferably accordingto the methods of Holliger et al. (Proc. Natl. Acad. Sci USA 90:6444,1993), incorporated herein by reference, to form diabody 38 which hastwo distinct antigen-binding regions 40 and 41, directed toward channel36 and a ligand 48, respectively. Ligand-binding region 40 consists of aheavy chain immunoglobulin 42 a and a light chain immunoglobulin 43 a.Ligand-binding region 41 consists of a heavy chain immunoglobulin 43 band a light chain immunoglobulin 42 b. Without intending to be bound byany particular theory, it is believed that the binding of binding region40 to one or more diabodies 38 to an epitope on cage 32 blocks channel36 and prevents substrate-enzyme interactions. Thus substrate 34 is notprocessed under non-stimulated conditions.

With reference to FIG. 2B, when binding region 41 attaches to ligand 48,the linker regions 44 and 46 of diabody 38 are distorted so that channel36 becomes unblocked and substrate 34 can pass through channel 36,indicted by arrow 49, and interact with enzyme 30. Once activated byligand 48, channel 36 remains open and multiple substrates 34 can enterand be acted on to produce products 34 a and 34 b which may be coloredproducts that are chromophoric or flourescent. Either one or bothproducts 34 a and 34 b are then detected or amplified, and may betransduced into a perceptible signal.

Although one skilled in the art would realize that various globularstructures, preferably proteins, could be used as cage 32,alpha-2-macroglobulin is particularly well suited for acting as anenzyme cage because several enzymes, including trypsin and thrombin,will partially degrade alpha-2-macroglobulin, enter the protein, andbecome entrapped merely by mixing the alpha-2-macroglobulin and enzyme.

Enzyme 30 can be any that can be entrapped in a molecular cage and thatproduces a detectable change of its substrate. One of ordinary skill inthe art would realize that a myriad of enzyme-substrate pairs aredetectable and therefore suitable. Thrombin and trypsin, for example,are two preferred enzymes. A preferred substrate has a recognition siteand a chromogen such that detectable colorimetric change occurs uponenzyme-substrate binding.

Linker regions 44 and 46 are engineered to allow substrate 34 to enterchannel 36 upon binding of ligand 48 to antigen binding region 41.Linker regions 44 and 46 are preferably of moderate length. The skilledartisan would appreciate that if the length of linker regions 44 and 46are too short or too long, then these regions may not distort adequatelyto unblock channel 36. The preferred range of linker region lengthdepends primarily on the nature of cage 32 and channel 36.

Linker regions 44 and 46 are also preferably bonded together by ionic,hydrogen, or other bonds. Otherwise, linker regions 44 and 46 would besusceptible to separation and consequently inadequate distortion uponbinding of a pathogenic agent.

An another embodiment of the present invention, representedschematically in FIGS. 3A-3C, also uses an enzyme-substrateamplification mechanism. As shown in FIG. 3A, a single chain chimericantibody 50 is engineered such that linker region 52 specifically bindsan enzyme 54 to inhibit the binding of a substrate 56 to enzyme 54, asindicated by arrow 58. In this embodiment, linker region 52 has aportion of its polypeptide sequence that is complementary to the activesite of enzyme 54. Techniques for the determination of suitablecomplementary polypeptides are well known.

As shown in FIG. 3B, when a binding region 60 of antibody 50 binds aligand 62, a conformational change in linker region 52 results in thedissociation of antibody 50 from enzyme 54. Liberated enzyme 54 is nowable to act on substrate 56. Each liberated enzyme can react withmultiple substrates to provide an amplified signal. By way of exampleonly, a particularly preferred embodiment is depicted in FIG. 3C showinghow an enzyme, such as luciferase, can be used to alter its substrate,in this example, the oxidation and dissociation of luciferin. Thisreaction produces a fluorescent species 57 a that is detectable belowpicogram levels of reaction product, and a non-fluorescent species 57 b.Fluorescent species 57 a is then detected or amplified and may betransduced into a perceptible signal. Enzyme 54 can be any enzyme thatproduces a detectable conversion of its substrate.

Experimental Liquid Crystal Amplification Mechanism

A ligand detection system using a liquid crystal amplification mechanismwas developed. A murine antibody to E. coli lipopolysaccharide (LPS), asurface antigen found on Gram-negative bacteria, was obtained from acommercial source (Biodesign International, cat. # C61212M, clone #26-5,lot #5D1197) and diluted (1:10) with sterile phosphate-buffered saline(PBS) pH 7.2 to yield a 100 ng/μl sample.

A late log phase culture of E. coli was grown in Brain Heart Infusion(BHI) broth and washed free of growth medium with 0.9% sterile saline.Bacterial numbers (determined by optical density at 600 nm andcolony-forming units (CFUs)) were quantitated by extrapolation fromgrowth curve data. Bacteria were then adjusted to 4.6×10¹ CFU per 5 μlby dilution with sterile saline.

The lyotropic liquid crystalline solution was prepared in a lamellarphase, using hexanol as a co-surfactant to help control the phase stateof the mixture. Cetylpiridiniumchloride (CpCl) was combined with hexanolin the proportion hexanol/CpCl=0.651 (w/w). The mixture was then dilutedwith a saline solution (1% NaCl in water) until 85% of the weight wassaline. The resulting liquid crystal was lamellar but close to themicellar phase (Nastishin, Langmuir. Vol. 12, pp. 5011-5015, 1996),incorporated herein by reference. In this phase, the lyotropic liquidcrystal is biphilic, and thus is capable of interacting with severaldifferent types of receptors.

The tested detection system was created by inserting a receptor(antibody) into the hexanol/CpCl/saline lyotropic liquid crystal. Thus,for each assay, 5 μl of the antibody (500 ng) solution, specificallyreactive against E. coli (LPS), was added to 5 μl of the lyotropicliquid crystalline solution and mixed. An experimental sample (5 μl) wasthen added to an aliquot (10 μl) of the liquid crystal-antibody mixtureand mixed. The experimental samples were added to the antibody-liquidcrystal mixture as follows: Sample A-5 μl of E. coli (4.6×10¹ CFU), aspecific bacteria that the system is designed to detect; Sample B-5 μlof PBS, or Sample C-5 μl (2×10⁶ CFU) S aureus, an irrelevant bacteriathat the system should not detect. The samples were centrifuged(3,500×g; 5 sec.) to eliminate bubbles, and 10 μl of the reactionmixture was placed on an ethanol-cleaned microscope slide, covered withan ethanol-cleaned glass cover slip, and the mixture was evaluated forbirefringence using polarized light. The experimental conditions andresults are summarized in Table 1. Birefringence occurred only when theantibody was bound to its specific antigen, with visually discernablechanges in birefringence detected for bacteria concentrations as low as46-460 CFU per 5 μl.

TABLE 1 Antibody Liquid Birefrin- Sample (LPS) E. coli S. aureus PBSCrystal gence A 500 ng 4.6 × 10¹ — — 5 μl YES B 500 ng — — 5 μl 5 μl NOC 500 ng — 2 × 10⁶ — 5 μl NO

In other experiments, LPS antibody, E. Coli (2.7×10⁷ CFU) and liquidcrystalline material were reacted in a manner similar to the previousexperiment, and representative photomicrographs (110× magnification;FIGS. 4A, 4B and 4C) were evaluated with a Bio-Quant Image AnalysisSystem. The image analysis was performed to quantatively comparepropagating light transmission when E. coli, PBS or S. aureus wasevaluated by the receptor-ligand binding system. The photographic imageswere digitized and integrated optical density (IOD) automaticallycalculated according to the following formula:

${IOD} = \frac{\left( {\sum{- \log_{10}}} \right)({foreground})}{({background})}$

The resulting data, presented in FIG. 5, show that a profound increasein transmission of propagating light occurs when liquid crystals amplifythe binding of antibody to E. coli (LPS).

Caged Enzyme Amplification Mechanism

The sensitivity of an alpha-2-macroglobulin amplification mechanism wasdemonstrated. Standard 2 mg/100 μl solution of humanalpha-2-macroglobulin (α₂M, Calbiochem Co., product number 441251),trypsin (Sigma Chemical Co., T-8003) and thrombin (Sigma Chemical Co.,T-4648) were made by dissolving the protease or antiprotease in 0.1 MHEPES buffer (pH 7.6). Equal volumes (100 μl) of α₂M and one of theenzymes were mixed, permitted to interact for ten minutes at roomtemperature and then cooled to 4° C. The 2001 sample was added to a gelfiltration column (1 cm×24 cm; 18.8 ml bed volume; 0.44 ml/min flowrate; located in a 4° C. cold room) packed with Sephadex G-100 toseparate the caged enzyme from uncomplexed enzymes. Column eluent wascollected in 1.0 ml fractions. Changes in light absorbance at 280 nm wasmeasured to determine the protein concentration in each fraction andenzymatic activity was determined to identify those fractions containingthe caged enzymes. The results of those measurements, shown in FIGS. 6Aand 6B, demonstrate that fractions 15-17 contained relatively puresamples of caged enzyme. Those fractions were used for the subsequentevaluations.

Small synthetic substrates, N-benzoyl-L-arg-p-nitroanalide andN-p-tosyl-gly-pro-arg-p-nitroanalide, were used to define the enzymaticactivities of caged trypsin (FIG. 6C) and caged thrombin (FIG. 6D),respectively. While both systems exhibited dose-responsecharacteristics, the caged thrombin exhibited greater sensitivity. Theenzymatic activity did not degrade with time. The enzymatic activity ofthe caged trypsin was unchanged following six days of storage at 4° C.(FIG. 6E). Similarly, caged thrombin activity was also stable whenmeasured 24 hours following preparation.

Luciferase Amplification Mechanism

The exceptional sensitivity of a luciferase-based amplificationmechanism was demonstrated using a Berthold Lumat Luminometer. Varyingamounts of luciferase (4 mg/ml of 0.15% NaCl, 10 mM HEPES, 1 mM EDTA, 2mM MgCl₂, 2 mM dithiothreitol; Sigma Chemical Co.) were added to theluminometer reaction chamber. The enzymatic reaction was initiated byrapid injection of 0.5 mM luciferin (Promega, E1483), 0.5 mM adenosinetriphosphate (Sigma Chemical Co., A-7699), 5 mM MgSO₄, 1.0 mMdithiothreitol (Sigma Chemical Co.) in 50 mM HEPES buffer (pH 7.8) intothe reaction chamber. FIG. 7B shows that detectable luciferase activitycan be measured with an enzyme concentration of only 0.0017 pg/μl, andlinear increases in activity are observed with progressive elevations inenzyme concentration.

It is to be understood that any variations evident fall within the scopeof the claimed invention, and thus the selection of specific antibodies,caged enzymes, receptor-inactivated enzymes or liquid crystals can bedetermined without departing form the spirit of the invention hereindisclosed and described. It should also be understood that the presentinvention, while particularly suited for pathogen detection, is intendedto include the detection of any ligand. Moreover, the scope of theinvention shall include all modifications and variations that may fallwithin the scope of the attached claims.

1. A device for the detection of ligands comprising: a liquid crystaldevice wherein a liquid crystal material is provided therein in apredetermined orientation to produce a first optical transmissionarrangement, a plurality of particles, each having at least one receptorcapable of binding to a ligand, mixed with the liquid crystal material,wherein upon binding of a ligand with the at least one receptor, thefirst optical transmission arrangement is varied; and a detector todetect the variation of the first optical transmission arrangementindicating detection of a ligand.
 2. The device of claim 1, wherein saidat least one receptor is attached to the surface of each of saidplurality of particles.
 3. The device of claim 1, wherein each of theplurality of particles is a porous substrate and said at least onereceptor is attached to at least one pore of a porous particle.
 4. Thedevice of claim 3, wherein a plurality of receptors are attached to andrandomly distributed on the surface and within the pores of said porousparticle.
 5. The device of claim 1, wherein the liquid crystallinematerial is selected from the group consisting of thermotropic liquidcrystalline material and lyotropic liquid crystalline material.
 6. Thedevice of claim 5, where the liquid crystalline material is a lyotropicliquid crystalline material.
 7. The device of claim 6, wherein thelyotropic liquid crystalline material is a lyotropic chromonic liquidcrystalline material.
 8. The device of claim 5, wherein the liquidcrystalline material is a thermotropic liquid crystalline material. 9.The device of claim 1, wherein the plurality of particles are made froma material selected from the group consisting of polymeric and inorganicmaterials.
 10. The device of claim 9, wherein the polymeric materialsare selected from the group consisting of polyions, polyalkenes,polyacrylates, polymethacrylates, polyvinyl, polystyrenes,polycarbonates, polyester, polyurethanes, polyamides, polyimides,polysulfones, polysiloxanes, polysilanes, polyethers, andpolycarboxylates.
 11. The device of claim 1, wherein the particles aresubstantially spherical.
 12. The device of claim 1, wherein said atleast one receptor is attached to each of said plurality of particles byat least one mechanism selected from the group consisting of (i)chemical attachment and (ii) physical attachment.
 13. The device ofclaim 12, wherein said chemical attachment is covalent bonding.
 14. Thedevice of claim 12, wherein said physical attachment is selected fromthe group consisting of: hydrophobic interactions and van der Waalsinteractions.
 15. The device of claim 1, wherein the at least onereceptor is an antibody.
 16. The device of claim 15, wherein theantibody is selected from the group consisting of monoclonal,polyclonal, and molecularly engineered antibodies.
 17. A device fordetecting ligands comprising: a plurality wells, each well containingthe device of claim
 1. 18. The device of claim 1, wherein the receptoris an antibody selected from the group consisting of monoclonal,polyclonal and molecularly engineered antibodies, wherein saidantibodies form a signal-producing receptor-ligand complex when thereceptor binds to the ligand.
 19. The device of claim 1, wherein theligand is a pathogenic agent.
 20. The device of claim 1, wherein thesignal is transduced into an optically perceptible signal.
 21. A devicefor the detection of ligand comprising: a plurality of particles havingat least one receptor attached to said particles, wherein said pluralityof particles are provided within a liquid crystalline material and saidat least one receptor is capable of binding to a ligand to form areceptor-ligand complex and wherein the formation of saidreceptor-ligand complex produces an optically detectable signal inrelation with the liquid crystalline material.
 22. A method fordetecting ligands comprising: providing a device capable of detectingligands, said device comprising a plurality of particles having at leastone receptor attached thereto, wherein said at least one receptor iscapable of binding to a ligand to form a receptor-ligand complex andwherein the formation of said receptor-ligand complex produces a signal;wherein the plurality of particles are disposed in a liquid crystallinematerial, exposing a sample containing at least one ligand to saiddevice allowing said receptor to interact with said at least one ligandto form at least one receptor-ligand complex, and measuring the signalproduced by said receptor-ligand complex formation.